Nature has devised a potent brake on the rate at which the pacemaker fires: signaling via the muscarinic cholinergic receptor (MR) pathway. The MR signaling cascade activated by vagal tone produces the most potent physiologic suppression of the rate of spontaneous electrical impulses generated within sinoatrial node, the primary heart pacemaker. Spontaneous beating rate reduction (BRR) of sinoatrial node cells (SANC) via MR signaling involves Gi protein coupling to several downstream targets. More specifically, signaling via Gi-beta-gamma activates IKACh, leading to membrane hyperpolarization; signaling via Gi-alpha inhibits adenylyl cyclase (AC) activity, leading to (a) a reduction in cAMP, a reduction of If current activation and changes in the early diastolic depolarization (DD), and (b) a reduction of cAMP-mediated, PKA-dependent activity. Since ICaL activation is controlled by PKA-dependent signaling, e.g. during beta-adrenoreceptors (beta-AR) stimulation, or in its absence, reduction of ICaL has been suggested as an additional BRR mechanism. Thus, it is currently believed that MR signaling slows heart rate entirely by the membrane-delimited mechanisms, including IKACh, If, and ICaL. On the other hand, recent studies showed that PKA also controls Ca2+ cycling (referred as Ca2+ clock in SANC) manifested as rhythmic spontaneous subsarcolemmal Ca2+ releases (LCRs) that affect the late diastolic depolarization (DD) via electrogenic Na+/Ca2+ exchange. A high basal PKA dependent phosphorylation of Ca2+ cycling proteins (phospholamban, ryanodine receptors, and L-type Ca2+ channels) is a crucial component of LCRs occurrence and is required for SANC spontaneous beating. While pharmacological inhibition of basal PKA activity damps LCRs resulting in BRR, there has been no prior investigation of how activation of MR signaling impacts on basal PKA signaling and functioning of the Ca2+ clock. Multiple mechanisms within sinoatrial nodal cells (SANC) can link muscarinic receptor (MR) activation to beating rate reduction (BRR). We examined the integration of mechanisms in response to a graded MR activation by Carbachol (CCh). The threshold CCh for BRR was 10[unreadable] nmol/l; the half maximal inhibition (IC50) was 100 nmol/l; and 1000 nmol/l abrogated beating. If blockade did not affect BRR at any CCh. Hyperpolarization, a marker of IKACh activation, became evident at CCh>30 nmol/l. During IKACh blockade (Tertiapin-Q, 1 mol/l) hyperpolarization did not occur, and at CCh>30 nmol/l the CCh-induced BRR was reduced in magnitude and slower to develop. At IC50 CCh reduced (1) adenylyl cyclase (AC) activity by 30% and PKA-dependent phospholamban phosphorylation by 40%; and (2) caused a timedependent slowing of spontaneous subsarcolemmal local Ca2+ release (LCR) periodicity that paralleled the concomitant time-dependent BRR. The dose response of BRR to CCh in the presence of IKACh blockade mirrored CCh effects on AC, phospholamban phosphorylation and LCR parameters. Pertussis toxin blocked all CCh effects on BRR. Numerical modeling validated experimental findings and demonstrated that Ca2+ cycling is integrated into MR modulation of BRR via LCR-induced Na+/Ca2+ exchanger current. Thus, MR stimulation-induced BRR is entirely dependent on Gi coupling to LCRs and IKACh but does not require If. At low CCh, i.e. in the absence of IKACh activation, BRR is attributable to a suppression of AC-phospholamban-Ca2+ signaling; as CCh increases beyond 30 nmol/l, a tight coupling between suppression of AC-phospholamban-Ca2+ signaling and IKACh activation underlies further stronger BRR.